Preparation of cationized gelatin nanospheres incorporating molecular beacon to visualize cell apoptosis

Abstract

The objective of this study is to prepare cationized gelatin nanospheres (cGNS) incorporating a molecular beacon (MB), and visualize cellular apoptosis. Two types of MB to detect the messenger RNA (mRNA) of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GAP MB), and caspase-3 (casp3 MB) were incorporated in cGNS, respectively. MB incorporated in cGNS showed the DNA sequence specificity in hybridization. The cGNS incorporation enabled MB to enhance the stability against nuclease to a significantly great extent compared with free MB. The cGNS incorporating GAP MB were internalized into the KUM6 of a mouse bone marrow-derived stem cell by an endocytotic pathway. The cGNS were not distributed at the lysosomes. After the incubation with cGNS, the cell apoptosis was induced at different concentrations of camptothecin. No change in the intracellular fluorescence was observed for cGNSGAPMB. On the other hand, for the cGNScasp3MB, the fluorescent intensity significantly enhanced by the apoptosis induction of cells. It is concluded that cGNS incorporating MB is a promising system for the visualization of cellular apoptosis.

Complexation design of cationized gelatin and molecular beacon to visualize intracellular mRNA

“…This can be due to the limited access of DNase I to the MB incorporated in the complexes as well as the inhibited recognition of MB as a substrate of the nuclease. In addition, it has been demonstrated that incorporation in the cationized gelatin nanospheres could prevent MB from the degradation by DNase I [ 30 ]. Therefore, in the case of complexes prepared in this study, the anti-nuclease stability would be enhanced compared with the free MB although further study is required to comprehensively investigate the incorporation ability of MB and the consequent stability for various complexes prepared with different cationized gelatins and mixing ratios.…”

“…The complexation of cationized gelatin and MB was performed by simply mixing cationized gelatin solution and MB in DDW [ 30 ] at different mixing ratios (5, 10, 20, 40, and 200 pmole MB/μg cationized gelatin). The mixed solution was incubated for 15 min at room temperature to obtain the cationized gelatin-MB complexes.…”

“…Molecular beacons (MB) composed of DNA bases for mouse messenger RNA (mRNA) of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enhanced green fluorescent protein (EGFP) were synthesized by Integrated DNA Technologies, Inc., Coralville, IA, USA. The sequences of MB [26,30] were as follows, GAPDH MB: 5'-[TYE 1 665]-CTGGTAATCCGTTCACACCGACCTTCACCAG-[Iowa Black 1 RQ]-3'; EGFP MB: 5'-[TYE 1 665]-ACGCCTTCTCGTTGGGGTCTTTGCTCGGCGT -[Iowa Black 1 RQ]-3' (underline indicates the stem structure). Target oligonucleotides of DNA for GAPDH MB (specific and non-specific sequences) were synthesized by Hokkaido System Science Co., Ltd, Sapporo, Japan.…”

“…MB was labeled with 125 I, as the methods previously reported [30]. Briefly, MB (50 μM, 5 μl) was incubated at 60˚C for 50 min with 2 μl of 0.3 mM Na 2 SO 3 , 5 μl of Na 125 I, and 5 μl of 4 mM PLOS ONE TlCl 3 .…”

Targeted Antitumor Mechanism of C-PC/CMC-CD55sp Nanospheres in HeLa Cervical Cancer Cells

“…Then the solutions were mixed with an appropriate amount of 1× binding buffer, and placed on ice. The samples were evaluated using flow cytometry within 1 h (Liang et al, 2017;Murata et al, 2018;Sui et al, 2018b;Zhao et al, 2018).…”